Method for the cytological analysis of cervical cells

ABSTRACT

The invention provides for a diagnostic test to monitor cancer-specific genetic abnormalities to diagnose cervical cell disorders and predict which patients might progress to cancer. Genetic abnormalities are detected by identification in chromosomal copy number of chromosome 3 and chromosome 5 using FISH analysis of probes targeted to 3q and/or 5p.

This Application is a continuing application of and claims priority toU.S. Ser. No. 12/506,985 which was filed on Jul. 21, 2009, which is anon-provisional of and claims priority to U.S. Ser. No. 61/082,346 whichwas filed on Jul. 21, 2008. The entire contents of Ser. No. 12/506,985are hereby incorporated by reference in their entirety.

FIELD

The invention relates to methods and kits for the analysis ofchromosomal abnormalities in cervical cells.

BACKGROUND

Cervical cancer is one of the most common and deadly cancers among womenworldwide. If detected early, cervical cancer and precursor lesions canbe treated effectively. A Pap test is the primary screen for cervicalcancer and uses morphological analysts to identify suspicious cells.However, a single cytologic examination is relatively insensitive,poorly reproducible and frequently yields equivocal results.Approximately 6% of Papanicolaou (Pap) tests are diagnosed as atypicalsquamous cells of undetermined significance (ASCUS) and requirefollow-up testing, and 5-10% of ASCUS patients have undetected cancer.Current guidelines for patients include follow-up Pap testing, testingfor human papilloma virus (HPV) and/or colposcopy.

Infection with HPV is associated with cervical cancer and many patientsare tested for HPV after an ASCUS Pap test result. The strength ofsensitive HPV testing is that it provides extremely high negativepredictive value; women who test negative are at low risk for developingcervical cancer. However, the positive predictive value of HPV testingis limited since only a small fraction of HPV positive early lesionsprogress to high-grade dysplasia and cancer. Thus, HPV detection, evenin combination with cytomorphological evaluation, is a test with poorspecificity.

In addition, approximately 3% of Pap tests are diagnosed with low-gradesquamous intraepithelial lesions (LSIL). Current guidelines for thesepatients recommend additional monitoring and/or colposcopy. Clinicalstudies show the majority of these patients are HPV+.

There is significant risk for an ASCUS/HPV+ or LSIL patient to progressto more severe cervical disease and require surgical treatment in thetwo years following the initial test. The identification of thesepatients that will progress is impossible based on morphology and HPVinfection. Genetic alterations have been identified in the earlydevelopment of cervical cancer that can predict the patient's risk ofdisease progression. These aberrations include gross changes in DNAcontent (e.g. ploidy) and the amplification of both a portion ofchromosome 3, specifically locus 3q26, that includes a gene TERC thatencodes a subunit the telomerase protein and a portion of chromosome 5,specifically 5p15, that includes a gene, TERT, that encodes anothersubunit of the telomerase protein, both of which are linked to cellimmortality. Studies have demonstrated multicolor fluorescent DNA probescan detect abnormalities in both ploidy, and 3q and 5p copy number byfluorescence in situ hybridization (FISH) with greater sensitivity andspecificity than other methods.

The implementation of cervical cancer screening programs has greatlyreduced disease incidence and mortality in industrialized countries.However, a single cytological evaluation remains relatively insensitive,hence the need for frequent follow-up investigations. This isattributable to sampling or interpretation errors, and to the fact thatsome early lesions may not have acquired recognizable phenotypicalterations. Invasive cervical carcinomas develop through increasingstages of cervical dysplasia, to cervical intraepithelial neoplasia(CIN) 1, CIN2, CIN3 and to carcinoma in situ, which is considered abonafide precancerous lesion that requires surgical intervention.However, only about 15% of all low-grade dysplastic lesions follow thispath of linear progression. Pap and HPV tests are indirect methods fordetermining the presence of cervical dysplasia or cancer. Therefore,there is a continuing unmet need for the methods of using molecularmarkers for directly identifying the presence of dysplasia or cancer andmonitoring disease progression.

SUMMARY

The invention provides for a diagnostic method to monitor geneticchanges in cervical cells using various cytological methods fordetecting hybridization, using FISH, CISH, flow cytometry, or othermethods as are known to those of skill in the art and for detectinggenetic abnormalities to predict which patients might progress to cancerand those unlikely to progress, months, if not years, before traditionalsymptoms present.

The visualization of chromosomal aneuploidy and copy number changes ofspecific cancer-associated genes has become an important complement toroutine morphological assessment of cytological samples. This approachis biologically valid and successful because chromosomal aneuploidy andthe resulting genomic imbalances are specific for cancer cells, distinctfor different carcinomas, and occur early during disease progression.Like most other human carcinomas, cervical cancers are defined by adistribution of genomic imbalances. In addition to infection withhigh-risk subtypes of human papilloma virus, the sequentialtransformation of cervical squamous epithelium requires the acquisitionof additional copies of chromosome arm 3q and 5p, among othercytogenetic abnormalities. In an aspect of the invention, identificationof a 3q26 amplification in addition to amplification of 5p15 in lowgrade cervical dysplasia can provide information regarding theprogressive potential of individual lesions to high grade cervicaldysplasia and cancer.

In one aspect, the present invention provides a method for assessing apatient condition of cervical cell disorder which may include cervicaldysplasia or cancer comprising: detecting, in a sample from a patient: agenomic amplification in chromosome 3q; a genomic amplification inchromosome 5p; and the presence and/or amplification of the centromereof chromosome 7 (CEN7) as control. Detecting the genomic amplificationof chromosome 3q and chromosome 5p indicates progression of the patientcondition to high grade cervical dysplasia. Defection of genomicamplification of chromosome 7 measures the general ploidy status of thecervical cell. Typically copy number changes in chromosome 7 do notoccur during early stage cervical carcinogenesis. If genomicamplification of chromosome 7 is present, it indicates aneuploidy, astate associated with advanced pre-cancers and cancers. The method canassess a change of patient condition of low grade cervical dysplasia toa condition of high grade cervical dysplasia or cancer.

In yet another aspect of the invention, a method for monitoring a shiftfrom a low grade to a condition of high grade cervical dysplasia in apatient is provided for. In certain aspects of the invention, themethods disclosed comprise assessing a change in a patient condition oflow grade cervical dysplasia to a condition of high grade cervicaldysplasia; identifying a patient at risk of developing invasive cervicalcarcinoma; and assessing maintenance of a patient condition of low gradecervical dysplasia or regression of a patient condition to low gradecervical dysplasia or normal.

In a specific aspect, the methods disclosed herein can further comprise,in addition to detecting genetic amplification in chromosome 3q and 5p,detecting amplification in chromosomes: 1q; 20q; 12q; 19q; 11q; 6q; 17p;7; 8q (detected in late stage dysplasia); 9q; 16q; 2q; 9p; 10q; 18p andany combination thereof. According to more specific aspects of theinvention, amplification in the 3q26 locus and 5p15 locus is detected.In addition to detection of the 3q26 and 5p15 loci, amplification in thefollowing chromosomal loci cans be detected: Iq21-31; 20q12; 12q13-24;19q13; 11q21; 7q11-22; 8q24 (detected in late stage dysplasia);9q33;-34; 16q23; 2q32; 9p22; 10q21-24; 18p11 and any combinationthereof.

The methods disclosed herein can further comprise determining that thegenomic amplification of chromosome 3q and/or chromosome 5p is presentin the sample or that the genomic amplification of chromosome 3q and/orchromosome 5p is not present in the sample.

In another aspect of the inventions, probes directed to chromosomalregions disclosed are provided, and kits are provided for conductingmethods of the invention.

These and other aspects of some exemplary embodiments will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following descriptions, while indicatingpreferred embodiments and numerous specific details thereof, are givenby way of illustration and not of limitation. Many changes andmodifications may be made within the scope of the embodiments withoutdeparting from the spirit thereof.

DESCRIPTION OF THE DRAWINGS

FIGS. 1 A, B and C illustrate the stages of cervical cancer progressionas represented by amplification of 3q26 chromosomal copy number gain.

FIG. 2 illustrates the stages of cervical cancer progression asrepresented by amplification of 3q26 and 5p15 chromosomal copy numbergain.

FIG. 3 illustrates the patient management process for cervical cancerdetection following Pap tests.

FIG. 4 is an illustration of negative results from a liquid-basedcytology patient sample testing for abnormality in 3q alone.

FIG. 5 is an illustration of positive results from a liquid-basedcytology patient sample testing for abnormalities in 3q alone.

FIG. 6 is an illustration of positive results from a liquid-basedcytology patient sample testing for abnormalities in 3q and 5p.

FIG. 7 is an illustration of negative results from a liquid-basedcytology patient sample testing for abnormalities in 3q and 5p.

FIGS. 8 A and B is an illustration of negative results from a patient'stissue biopsy sample testing for abnormalities in 3q alone. FIG. 8A isfrom a CIN1 tissue biopsy. FIG. 8B is from a CIN2 biopsy.

FIGS. 9 A, B and C is an illustration of positive results from apatient's tissue biopsy sample testing for abnormalities in 3q alone.FIG. 9A is from a CIN1 tissue biopsy. FIG. 9B is from a CIN2 tissuebiopsy. FIG. 9C is from a CIN3 tissue biopsy.

FIG. 10 is an illustration of results from a patient's tissue biopsysample testing for abnormalities in 3q and 5p.

FIG. 11 is an illustration of a liquid-based cytology patient sampletesting for abnormalities in 3q alone with tetraploid cells.

FIG. 12 is an illustration of a liquid-based cytology patient sampletesting for abnormalities in 3q and 5p with tetraploid cells.

FIG. 13 illustrates genomic organization of chromosomal locus 3q26,including the gene position of TERC, among other genes, and BAC clonessuitable for the production of labeled DNA probes.

FIG. 14 illustrates genomic organization of chromosomal locus 5p15,including the gene position of TERT and TRIP13, among other genes, andBAC clones suitable for the production of labeled DNA probes.

FIG. 15 illustrates clone order used to identify probes in band 5p15.33and specific and BAC clones suitable for the production of labeled DNAprobes to TRIP 13.

FIGS. 16 A, B, C, and D illustrate a listing of genes that can betargeted by specific probes of the invention to measure chromosomalabnormalities in cervical cells.

DETAILED DESCRIPTION

The present invention is based on the identification of gain in copynumber of chromosomal regions associated with cervical cancer. Cancer isa genetic disease, and genetic aberrations can be observed in diseasedcells. The aberrations can be observed cytologically, by measuringgenetic aberrations either as increase or decrease in gene regions.Also, certain gene expression differences are evident in cancer cellssuch that measurement of biomarker expression can be a diagnosticindicator of disease state in the cell, whether or not it can beobserved cytologically. The methods discussed herein can directlyidentify abnormalities in the DNA of cervical cells using fluorescentlylabeled probes that bind to the aberrant regions in the chromosome. Whengreater than, or less than, the expected number of signals are observed,a cell sample can be diagnosed as diseased and cervical dysplasia can bediagnosed before it can be observed cytologically. Patients with theseabnormalities can have a poor prognosis and can be at high risk todevelop more advanced cervical disease. The methods disclosed herein canbe performed subsequent to or in lieu of ASCUS/HPV+ or LSIL Pap tests,among other abnormal results from cytology testing, in order to providemore specific information about a patient's risk of disease progression.

As used herein, “cervical cell disorder,” “cervical disorder,” or“cervical disease” means any of the following: cervical carcinogenesis,Negative for Intraepithelial Lesion or Malignancy (NILM), HumanPapilloma Virus (HPV) positive, Atypical Squamous Cells of UndeterminedSignificance (ASC-US), Low-grade Squamous Intraepithelial Lesion (LSIL),Atypical Squamous Cells, HSIL (ASC-H), Atypical Glandular Cells ofUndetermined Significance (AGUS), High-grade Squamous IntraepithelialLesion (HSIL), cervical dysplasia, pre-cancer, pre-malignant legion,cervical cancer, cervical adenocarcinoma, cervical squamous cellcarcinoma, cervical intraepithelial neoplasia 1 (CIN1), cervicalintraepithelial neoplasia (CIN2), cervical intraepithelial neoplasia 3(CIN3), carcinoma in situ, invasive cervical carcinoma, and cytologicalor genetic abnormality of the cell. Also, “disease,” “cell disorder,” or“disorder” as used herein includes but is not limited to any cytologicalor genetic abnormality of the cell.

The present method provides direct identification of geneticabnormalities in morphologically normal cells and abnormal cells, aswell as prognostic information about disease progression, and theflexibility to work with both squamous and glandular cervical cells.

Copy Number Gains

An increase in 3q copy number, in addition to integration of humanpapilloma virus (HPV) into the host genome, have been associated withthe progression of CIN2 or CIN 3 to cervical carcinoma such that bothappear to be important associated events in the progression of cervicaldysplasia to invasive cancer. Hopman et al. J. Pathol. 2006 December:210(4): 412-9. Higher staged tumors or those with lymph node metastasishad more chromosomal imbalances including gains of 3q; 1q; 8q; andlosses of 11q; 3q; 6q and 2q. Gains of 3q11-q22 and 3q26-qter were moreprevalent with lymph node metastasis. Huang, K. F. et al., J. Formos MedAssoc. 2007 November; 106(11): 894-902.

3q gains seen in invasive cervical carcinomas, specifically gain in thehuman telomerase gene (TERC), have been used in the development of FISHprobe sets as a diagnostic tool in the detection of TERC gains in Papsmears. It has been suggested that TERC gains could predict progressionfrom CIN1/CIN2 to CIN3 and invasive carcinoma. Heselmeyer-Haddad et al.Am Journal of Pathology 2005; 166:1229-1238.

5p is also a frequently observed structurally changed chromosome incarcinomas. Atkin, N. B 1997 Elsevier; 95: 33-39. Arias-Pulido, H. etal. 2002 Mol. Cancer; 1:3. Huang F. Y., et al. 2005 Cancer Gen. andCyto., 157: 46-47, Macville M., et al. 1999 Cancer Res.; 59:141-50.Heselmeyer K. et al. 1997 Genes Chromosomes Cancer; 19: 233-40. Rao P.H. et al. 2004 BMC Cancer; 4:5. 5p gains are observed during progressionto advanced stage carcinomas, and frequently involve whole armamplifications. Heselmeyer K. et al. 1997 Genes Chromosomes Cancer; 19:233-40.

Using carcinoma cell lines that showed 5p amplification, a minimalregion of alteration at 5p13.33 has been defined, which encodes thehuman telomerase reverse transcriptase (hTERT) gene. Lockwood, W. et al.Int. J. Cancer 2006; 120: 436-443. Finally, an HPV integration site hasalso been mapped to 5p11-15. Lockwood, W. et al. Int. J. Cancer 2006;120: 436-443. Telomerase activation is a component of cancer cellimmortality Takuma, Y. et al. 2004 Journal of Gastroenterology andHepatology; 19; 1300-1304. Takahashi S. et al. 2000 Eur. Jour. OfCancer, 36: 496-502. Toshikuni, N. et al. 2000 Br. J. Cancer; 82;833-837. hTERT has been identified as the catalytic subunit oftelomerase. Takahashi S. et al. 2000 Eur. Jour. Of Cancer, 36; 496-502.

hTERT expression has been observed in several cancer cell lines,including cervical carcinomas, with certain cancer cell lines showingthat hTERT expression is high in cancerous lesions but not non-canceroustissues. Takahashi S. et al. 2000 Eur. Jour. Of Cancer, 36: 496-502.Although this differential expression was found in hepatocarcinomasrather than cervical carcinomas, the results suggest that hTERTexpression occurred at an early stage of hepatocarcinogenesis. TakahashiS. et al. 2000 Eur. Jour. Of Cancer, 36: 496-502. Further, 5p hTERT geneamplification is closely correlated with increased hTERT mRNA expressionin cervical cancers with HPV infection. Zhang A. et al. 2000 CancerRes.; 60: 6230-6235. Zhang A. et al. 2002 Genes Chromosomes Cancer; 34:269-75. 5p has been consistently identified as a chromosome thatundergoes structural changes during various stages of carcinogenesis.The structural changes also appear to consistently affect the TERT geneencoded on 5p.

5p gain has been observed in invasive cervical carcinoma. Scotto, etal., Molecular Cancer 2008. When observed in samples in addition toobservations of gain in 3q, specifically 3q26, it can be an indicator ofincreased progression of disease state from cervical dysplasia toinvasive cervical carcinoma. Using a genomic probe for a region on 3q,specifically chromosome band 3q26, in combination with at least onecontrol probe, (eg. CEP3, CEN7), and a genomic probe for a region on 5p,especially 5p15, the copy number increases precede malignant conversionof cervical intraepithelial neoplasms to invasive carcinoma, and furtheraccompany the transition from ASCUS or CIN1 to CIN2 or CIN3 and fromCIN2 or CIN3 to carcinoma in situ to invasive cervical carcinoma.Moreover, the identification of gain in both 3q and 5p indicates anexpedited transition from ASCUS or CIN1 to or CIN3 and from CIN2 or CIN3to carcinoma in situ to invasive cervical carcinoma.

The present methods provide for identification of possible cervical celldisorder by comparing the copy number increase of the targetchromosomes, for example, 5p, 3q or both together, as compared tonormal. As used herein, “normal” means chromosomal diploidy in mammaliancells except when cells that are normally diploid are tetraphase and inthe cell cycle and tetraploidy is observed.

All cells have a normal complement of 23 pairs of chromosomes a statethat is described as diploid. However, when cells grow and undergo celldivision they generate a second set of 23 pairs of chromosomes one setwill subsequently reside in the two daughter cells that are created.This state is described as tetraploid. While tetraploidy is a naturalprocess that occurs throughout the body's tissues and organs on aregular basis, it occurs at low frequency, in general. One hallmark ofcancer is uncontrollable cell growth and replication. This typicallyoccurs due to multiple abnormalities in the chromosomes of the cell thatenable the cell to escape the standard replication control systemswithin normal cells. These multiple abnormalities within the chromosomeslead to a state described as aneuploidy, where the chromosome complementis no longer 23 pairs, but something else. Typically, aneuploid cellshave extra copies of some chromosomes, have lost other chromosomes, andhave even created hybrid chromosomes by fusing two or more together.Very active cell division and tetraploidy provides a foundation foraneuploid cells to develop. Tetraploid can, therefore, be a transitorycondition that indicates a higher risk level for the development ofaneuploid cells and more severe cell disorders. Therefore, these methodscan measure tetraploidy and provide for the identification of cervicalcell disorder and possible progression to cervical cancer according tothe methods disclosed herein.

Methods

The methods can be used as a diagnostic and prognostic marker forcervical dysplasia. Patients with ploidy abnormalities and/or increased3q and/or increased 5p copy numbers have a poor prognosis and are athigh risk to develop more advanced cervical disease.

It is an embodiment of the present invention to identify changes in DNAcontent and 3q+5p copy number in cervical cytology samples usingmulticolor FISH probes directed to loci on chromosomes 5p and 3q anddirected to CEN7, more specifically, the probes are directed to 5p15 and3q26. In a preferred embodiment, probes to different targets willfluoresce with a different color so that targets can be differentiated.

It is an embodiment of the present invention to provide a method forassessing a patient condition of cervical dysplasia or cancer,comprising, detecting in a sample from a patient: a genomicamplification in chromosome 3q; a genomic amplification in chromosome5p; the presence and/or amplification of CEN7. Detecting the genomicamplification of chromosome 3q and chromosome 5p indicates progressionof the patient condition from low grade to high grade cervicaldysplasia.

The methods of the invention can be used to monitor a shift from a lowgrade to a condition of high grade cervical dysplasia in a patientsample: a genomic amplification in chromosome 3q; a genomicamplification in chromosome 5p; the presence and/or amplification ofCEN7. Detecting the genomic amplification of chromosome 3q andchromosome 5p indicates progression of the patient condition to highgrade cervical dysplasia.

The methods disclosed herein may further comprise, in addition todetecting genetic amplification in chromosome 3q and 5p, detectingamplification in chromosomes; 1q; 20q; 12q; 9q; 11q; 6q; 17p; 7; 8q(detected in late stage dysplasia); 9q; 10q; 2q; 9p; 10q; 18p and anycombination thereof.

According to specific embodiments of the invention, amplification, inthe 3q26 locus and 5p15 locus band, including the Cri du Chat region,can be detected. In yet further specific embodiments of the invention,in addition to detection the 3q20 and 5p15 loci, amplification in thefollowing chromosomal loci can be detected: 1q21-31; 20q12; 12q13-24;19q13; 11q21; 7q11-22; 8q24 (detected in late stage dysplasia); 9q33-34;16q23; 2q32; 9p22; 10q21-24; 18p11 and any combination thereof.

The method can further comprise determining that the genomicamplification of chromosome 3q and/or chromosome 5p is not present inthe sample. When compared to a state where aneuploidy is found andcervical cell disorder is identified, the loss of chromosomalamplification can be indicative of regression of cell disorder andpossibly regression of disease.

The methods may be used for assessing and monitoring late stagedysplasia comprising detecting genomic amplification in chromosomes 3q,as well as 5p and 8q, more specifically 8q24. Gain of 8q copy numberand/or gain in 5p copy number in combination with gain in 3q, canindicate malignant conversion of cervical intraepithelial neoplasms toinvasive carcinoma, and further accompany the transition from ASCUS/CIN1to CIN2/CIN3 and from CIN2/CIN3 to carcinoma in situ to invasivecervical carcinoma. Moreover, the identification of gain in both 3q and5p indicates an expedited transition from ASCUS/CIN1 to CIN2/CIN3 andfrom CIN2/CIN3 to carcinoma in situ to invasive cervical carcinoma.

The methods further provide for a specific probe panels including probesto chromosomes 3q and 5p, and can further include probes to: 1q; 29q;12q; 19q; 11q; 6q; 17p; 7; 8q (detected in late stage dysplasia); 9q;16q; 2q; 9p; 10q; 18p and any combination of probes thereof. Accordingto specific embodiments of the aforementioned probe panel, probes to the3q26 locus and 5p15 locus, including the Cri du Chat region, in additionto, probes to the following chromosomal loci can be used: 1q21-31;20q12; 12q13-24; 19q13; 11q21; 7q11-22; 8q24 (detected in late stagedysplasia); 9q33;-34; 16q23; 2q32; 9p22; 10q21-24; 18p11 and anycombination thereof.

One of skill in the art can prepare nucleic acid probes that arecomplimentary to the sequences of the loci described herein.Additionally, many such probes are commercially available.

Recent clinical research has illuminated the role of HPV infections isthe development of anal cancers. In fact, the Centers for Medicare andMedicaid Services (CMS) recently began providing coverage for a range ofcytology and HPV testing of anal specimens. Because of the similarbiology between cervical and anal carcinogenesis, including similar celltypes and viral initiation, genomic abnormalities and copy numberchanges occur at 3q and 5p among other loci. It is therefore a furtherembodiment of the present invention to analyze anal cell specimens for3q and 5p among the other chromosomal copy number changes to determinewhether a patient may have anal disease. Therefore, these methods couldbe used on anal specimens and provide valuable clinical informationregarding anal carcinogenesis.

Probes

A number of methods can be used to identify probes which hybridizespecifically to the specific loci exemplified herein. For instance,probes can be generated by the random selection of clones from achromosome specific library, and then mapped by digital imagingmicroscopy. This procedure is described in U.S. Pat. No. 5,472,842.Various libraries spanning entire chromosomes are also availablecommercially from for instance Illumina Inc. Probes that hybridizespecific chromosomal loci are available commercially from AbbotMolecular, Inc. (Des Plaines, Ill.)

Briefly, a genomic or chromosome specific DNA is digested withrestriction enzymes or mechanically sheared to give DNA sequences of atleast about 20 kb and more preferably about 40 kb to 300 kb. Techniquesof partial sequence digestion are well known in the art. See, forexample Perbal, A Practical Guide to Molecular Cloning, 2nd Ed. WileyN.Y. (1998). The resulting sequences are ligated with a vector andintroduced into the appropriate host. Exemplary vectors suitable forthis purpose include cosmids, yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs) and P1 phage. Various librariesspanning entire chromosomes are also available commercially from forinstance Genome Systems.

Once a probe library is constructed, a subset of the probes isphysically mapped on the selected chromosome. FISH and digital imageanalysis can be used to localize clones along the desired chromosome.Briefly, the clones are mapped by FISH to metaphase spreads from normalcells using e.g., FITC as the fluorophore. The chromosomes may becounterstained by a stain which stains DNA irrespective of basecomposition (e.g., DAPI or propidium iodide), to define the outlining ofthe chromosome. The stained metaphases are imaged in a fluorescencemicroscope with a polychromatic beam-splitter to avoid color-dependantimage shifts. The different color images are acquired with a CCD cameraand the digitized images are stored in a computer. A computer program isthen used to calculate the chromosome axis, project the two (for singlecopy sequences) FITC signals perpendicularly onto this axis, andcalculate the average fractional length from a defined position,typically the p-telomere. This approach is described, for instance, inU.S. Pat. No. 5,472,842.

Sequence information of the genes identified here permits the design ofhighly specific hybridization probes or amplification primers suitablefor detection of target sequences from these genes. As noted above, thecomplete sequence of these genes is known. Means for detecting specificDNA sequences within genes are well known to those of skill in the art.For instance, oligonucleotide probes chosen to be complementary to aselected subsequence within the gene can be used. Alternatively,sequences or subsequences may be amplified by a variety of DNAamplification techniques (for example via polymerase chain reaction,ligase chain reaction, transcription amplification, etc.) prior todetection using a probe. Amplification of DNA increases sensitivity ofthe assay by providing more copies of possible target subsequences. Inaddition, by using labeled primers in the amplification process, the DNAsequences may be labeled as they are amplified.

In one embodiment probes of the present invention may be directed to atleast a portion of TERC gene at band 3q26.2 and TERT or TRIP13 at5p15.3. Specifically, a probe to TERC at region 3q26 of approximately495 kb can be used labeled with spectrum gold and also a probe for 5p15labeled with spectrum green. Such probes are commercially available fromAbbot Molecular (Des Plaines, Ill.). However, the probes of theinvention can include any gene on the 3q26 and 5p15 including genes inthe Cri du Chat region and those listed in FIGS. 16A-D and anycombination or portion of the genes on 3q26 or 5p15.

In a specific embodiment, the detectable marker of the probe can emit afluorescent signal or the probe may be chromogenic. The probes arehybridized using fluorescent in situ hybridization (FISH). FISH is acytogenetic technique used to detect or localize the presence or absenceof specific DNA sequences on chromosomes. FISH uses fluorescent probesthat bind to parts of the chromosome with which they show a high degreeof sequence similarity. Fluorescence microscopy can be used to find outwhere the fluorescent probe binds to the Chromosome. In situhybridization is a technique that allows the visualization of specificnucleic acid sequences within a cellular preparation. Specifically, FISHinvolves the precise annealing of a single stranded fluorescentlylabeled DNA probe to complementary target sequences. The hybridizationof the probe with the cellular DNA site is visible by direct detectionusing fluorescence microscopy.

In instances where additional genetic material is required for testing,the genome may be amplified or detected by Polymerase Chain Reaction(PCR).

It is yet another embodiment of the invention to provide for a procedureof performing FISH on liquid cytology specimens such as SUREPATH® orTHINPREP® specimens for successful hybridization of DNA probes inpracticing the methods disclosed herein. SUREPATH® is available fromBecton-Dickinson of Sparks, Md. THINPREP® is available from HologicLaboratories of Bedford, Mass.

It is yet another aspect of the invention to use antibodies to separatesquamous and glandular cells out of liquid-based cytology specimensprior to defecting genetic amplification in sample cells. The separationof cell types can improve detection of both squamous and glandularcancers and improve detection of cervical carcinomas which are rarelydetected through traditional Pap testing but show 3q26 amplification,5p15 amplification, or both.

The present methods can utilize probes that are fluorescently labelednucleic acid probes for use in in situ hybridization assays. The labeledprobe panel may consist at least of a three-color, three-probe mixtureof DNA probe sequences homologous to specific regions on chromosomes 3,5 and 7; and, as well as other chromosome regions disclosed herein.

As used herein “label” or “labels” is any composition, e.g. probe,detectable by spectroscopic, photochemical, biochemical, immunochemical,or chemical means including but not limited to fluorescent dyes (e.g.fluorescein, rhodamine, Texas Red, etc., enzymes, electron densereagents, magnetic labels, and the like). Labels which are not directlydetected but are detected through the use of indirect label includebiotin and dioxigenin as well as haptens and proteins for which labeledantisera or monoclonal antibodies are available. Methods of labelingnucleic acids and probes are well known to those of skill in the art.Preferred labels are those that are suitable for use in in situhybridization. The nucleic acid probes may be detectably labeled priorto hybridization. Alternatively, a detectable label which binds to thehybridization product may be used. Such detectable labels include anymaterial having a detectable physical or chemical property and are welldeveloped in the field of immunoassays.

It is yet another embodiment of the present methods whereby squamousand/or glandular cervical cells can be used from a patient sample toassess chromosomal abnormalities using the present methods.

Typically, it is desirable to use multiple color, in a preferredembodiment three-color FISH methods for detecting chromosomalabnormalities in which three probes are utilized, each labeled by adifferent fluorescent dye. In the preferred embodiment, two test probesthat hybridizes to the regions of interest are labeled with twodifferent dyes and a control probe that hybridizes to a different regionis labeled with a third dye. More than three probes can be used so longas each is labeled with a unique dye. A nucleic acid probe thathybridizes to a stable region of the chromosome of interest such as thecentromere, is preferred as a control probe so that differences betweenefficiency of hybridization from sample to sample can be determined.

Cells recovered and isolated from specimens or samples collected frompatients can be fixed on slides. Specimens can be retrieved usingvarious techniques known in the art. In one embodiment, specimens can beretrieved from THINPREP® and/or SUREPATH® samples. SUREPATH® is a Paptest used for the screening of cervical cancer. SUREPATH® has variouscollection devices to collect Pap samples from a patient. Some havedetachable heads that hold the sample, are directly detached and putinto a vial that is sent for screening, enabling 100% of sample to beavailable for processing. A liquid-based Pap test using thin-layer cellpreparation process called the BD SUREPATH® liquid-based Pap test whichclaims an increase in detection rate compared to the conventional Papsmear is used with the SUREPATH® collection devices such as thebroom-like device or the brush/spatula with detachable heads, asdisclosed U.S. patent application Ser. No. 11/521,144, incorporatedherein by reference in its entirety. The THINPREP® Pap is a liquid-basedcytology method. A sample of the cervical cells is rinsed into a vialinstead of a smear onto a slide thus preventing clumping of cells. Thecells are separated in a laboratory to eliminate blood and mucus and thecells to be studied are then placed on a slide for studies to detectcancerous cells.

The samples may also comprise analysis of tissue from cervical biopsies,punch biopsies, surgical procedures including LEEP, hysterectomy, CONEbiopsy, ECC. The sample may be prepared from tissue or cells removedfrom the cervix, vagina or vulva.

Hybridization

In an embodiment, the regions disclosed here are identified using insitu hybridization. Generally, in situ hybridization comprises thefollowing major steps: (1) fixation of tissue or biological structure tobe analyzed; (2) pre-hybridization treatment of the biological structureto increase accessibility of target DNA, and to reduce nonspecificbinding; (3) hybridization of the mixture of nucleic acids to thenucleic acid of the biological sample or tissue; (4) post-hybridizationwashes to remove nucleic acid fragments not bound in the hybridizationand (5) detection of the hybridized nucleic acids. Hybridizationprotocols for the applications described herein are described in U.S.Pat. No. 6,277,563, incorporated herein by reference in its entirety.

From samples, the target DNA can be denatured to its single strandedform and subsequently allowed to hybridize with the probes of themethod. Following hybridization, the unbound probe is removed by aseries of washes, and the nuclei are counterstained with DAPI (4, 6diamidino-2-phenylindole), a DNA-specific stain. Hybridization of theDNA probes can be viewed using a fluorescence microscope equipped withappropriate excitation and emission filters allowing visualization ofthe aqua and gold fluorescent signals. Enumeration of CEN 7, 5p15 and3q26 signals is conducted by microscopic examination of the nuclei.

The clinical test disclosed herein can use several biomarkers incombination for the early detection of cervical cancer and is importantbecause current morphology based screening and detection methods havesignificant limitations. Identification of 3q26 and 5p15, among others,amplification and other cytogenetic abnormalities can more precisely andaccurately identify patients at risk for developing cervical cancer andhelp them receive earlier treatment.

Image Analysis

It is an embodiment of the present invention to provide for automaticimage analysis and scoring of the methods disclosed. In situhybridization is a technique that allows the visualization of specificnucleic acid sequences within a cellular preparation. Specifically, DNAfluorescence in situ hybridization (FISH) involves the precise annealingof a single stranded fluorescently labeled DNA probe to complementarytarget sequences. The hybridization of the probe with the cellular DNAsite is visible by direct detection using fluorescence microscopy. Themethod, as described herein, utilizes probes that are fluorescentlylabeled nucleic acid probes for use as part of in situ hybridizationassays. In a preferred embodiment, the probe panel consists of a3-color, three-probe mixture of DNA probe sequences homologous tospecific regions on chromosomes 3, 5, and 7. The probe mixture consistsof a locus specific probe for chromosome 3q26, 5p15, and centromere ofchromosome 7 (CEN 7).

It is an embodiment of the present invention to provide for automatedimage analysis of the signal from the FISH probe. Microscopes can allowfor automated capture of digital images of the field of view within thespecimen/slide on the microscopy stage. Such manufacturers include CarlZeiss, Leica, Nikon and Olympus. Also, the method provides for softwareplatforms for automated image analysis such as microscope-softwaresystems developed by such entities as Ikonisys of Connecticut,Metasystems of Massachusetts and Germany and Bioimagene of California,Bioview of Massachusetts, and Israel, among others. Such automatedsystems may apply to viewing 3q chromosomes alone or in combination with5p abnormalities in the patient sample.

Cells recovered from specimens can be fixed on slides. The target DNA isdenatured to its single stranded form and subsequently allowed tohybridize with the probes. Following hybridization, the unbound probecan be removed by a series of washes, and the nuclei are counterstainedwith DAPI (4,6 diamidino-2-phenylindole), a DNA-specific stain.Hybridization of the probes can be viewed using a fluorescencemicroscope equipped with appropriate excitation and emission filtersallowing visualization of the three fluorescent signals. Enumeration ofCEN7, 5p15 and 3q26 signals is conducted by automated microscopicexamination of the nuclei.

The probe set and DAPI countersign can be viewed on an epi-fluorescencemicroscope equipped with a 100-watt mercury lamp equipped with thefollowing filters: DAPI, Spectrum Aqua (chromosome 7 centromere),Spectrum Green (locus on 5p15), and Spectrum Orange (locus on 3q26) orother labels and probes as are known in the art and disclosed herein.DAPI filter and a magnification of 100× can quickly scan sample area ofpatient slide to determine cell quantity and quality. Analysis begins inthe upper left quadrant of the target area. Scan fields with 63× oilobjective from left to right and top to bottom, without re-scanning thesame areas. The system can count a total of 1000 cells. The methodfurther comprises automatic scoring of the cell counts.

Clinical Significance of Slide Analysis Procedure:

While the performance of FISH laboratory procedures on specimens can bechallenging, the resulting procedural analysis needs to be placed withincontext of the disease and the relevance of the technical results toclinical practice must be determined. The method disclosed herein isdesigned to be a direct evaluation of chromosomal copy number atspecific loci associated with cervical cell disorders. The presence ofthese genetic abnormalities in cervical cancer screening specimens, suchas a Pap test, long before the development of cancer has implicationsfor the management and treatment of patients. The results of aFISH-based analysis of the specimen can be considered within clinicalcare guidelines and procedures.

Determination of chromosomal copy number in at least 800 cells, andpreferably 1000 cells, can be a sufficient sampling of each clinicalspecimen. Less than 800 cells or more than 1000 cells can be utilized inthis system. The method and system overcome sampling variations andlimitations of slide production methodology. The methods and system areconsistent with methods recommended by professional medicalorganizations (ACMG) to determine the threshold between a specimen withand without chromosomal copy number changes. Wolf, D. J. et al. (2007)Period Guidelines for Fluorescence In Situ Hybridization Testing.

The automated method and system provides for at least 90% accuracy forpositive specimens and identifies a patient with an increased risk ofdisease progression. The method and system can further provide forgreater than 95% accuracy.

In situ hybridization is a technique that allows the visualization ofspecific nucleic acid sequences within a cellular preparation.Traditionally the visualization of probe signals has been performedmanually by highly-trained personnel. However, it is possible to adaptcurrent technology to automate the image acquisition and analysisprocess. Microscopes on the market today, such as those manufactured byCarl Zeiss, Leica, Nikon, and Olympus, allow the user to capture digitalimages of the field of view within the specimen/slide on the microscopystage. Some of these manufacturers have software available for theautomated acquisition of images from specimens/slide. In addition,several entitles (Ikonisys, Metasystems, Bioimagene, BioView, Aperio,Ventana, among others) have created software platforms specifically foruse in commercial laboratories. Some of these entities have systems thatinclude both a microscopy platform and the automated imaging software,including the Ikoniscope Digital Microscopy System by Ikonisys andMetafer and Metacyte by Metasystems.

The type and source of the specimen to be analyzed directly impacts theanalysis process and methodology. Each tissue type has its own biologyand structure plus each cancer develops differently with differentfactors affecting the rate of carcinogenesis. Therefore the presentinvention provides for several methods for automated image acquisitionand analysis of specimens.

It is an embodiment of the system and method to be used in conjunctionwith specimens in liquid suspension that can be placed onto a microscopeslide in an even, monolayer of cells, this includes liquid-base cytologyspecimens such as THINPREP® and SUREPATH® plus any fine-needle aspirate(FNA), sputum, or swab-based collection. This automated method screensthe entire area covered by cells on the FISH prepared slide and utilizesthe DAPI-stain to identify cellular nuclei. The system then enumerateseach probe signal within the DAPI-stained region and records the copynumber of each probe identified. The software system continues itsautomated scoring of cells and chromosomal copy number within each celluntil it obtains results of at least 800 cells. Once the 800 cellthreshold is reached, the software can categorize each cell imaged andcounted into a category based upon the copy number of each chromosomeidentified. A normal cell with two copies of each probe 3q26, 5p15, andCEN7 would be placed into a 2,2,2 category. Abnormal cells would beidentified by their probe signal patterns. For instance, a cell with twocopies of the CEN7 probe, 5 copies of the 3q26 probe and 3 copies of the5p15 probe can be placed in the 2,5,3 category. Once all of the imaged,cells are categorized, the specimen can be evaluated relative to thepositive/negative disease threshold. All cells identified as abnormal bythe automated imaging system can be reviewed and verified manually bytrained personnel before test results are communicated to a physician.The method and system further provides for automated verification.Specific cell threshold numbers can vary by specimen type and collectionmethod. In addition, the software can be adapted to reflect biological(cell shape, cell size, DNA content of the nucleus, proximity of cellsto each other, cell type, etc.) or disease related differences (numberof loci with abnormal number, the number of abnormalities at a locuswithin a single cell, relationship of an abnormality to survival ortreatment response). This method and system can be used on arepresentative sampling of area covered by cells on the slide instead ofthe entire area, typically this is performed by imaging multiple fieldsof view or a path based on cellular density until the minimum imagedcell threshold is met.

It is yet a further embodiment of the system and method that it can alsobe used in conjunction with specimens in liquid suspension that canplaced onto a microscope slide in an even, monolayer of cells, thisincludes liquid-base cytology specimens such as THINPREP® and SUREPATH®plus any fine-needle aspirate (FNA), sputum, or swab-based collection.This automated method screens the entire area covered by cells on theFISH prepared slide and utilizes the DAPI-stain to identify cellularnuclei. The system then enumerates each probe signal within theDAPI-stained region and records the copy number of each probeidentified. The software system continues its automated scoring of cellsand chromosomal copy number within each cell until it obtains results ofall of the cells on the slide. Once all of the cells are imaged, thesoftware categorizes each cell imaged and counted into a category basedupon the copy number of each chromosome identified. For instance, anormal cell with two copies of each probe 3q26, 5p15, and CEN7 would beplaced into a 2,2,2 category. Abnormal cells would be identified bytheir probe signal patterns. For instance, a cell with two copies of theCEN7 probe, 5 copies of the 3q26 probe and 3 copies of the 5p15 probewould be placed in the 2,5,3 category. Once all of the cells are imaged,counted and categorized, the software identifies the cells with thegreatest number of abnormalities and provides a descending rank-basedordering of the highly abnormal cells. This rank-based ordering of thehighly abnormal cells within the specimen can be evaluated relative tothe positive/negative disease threshold. Typically, but not always,cells identified as abnormal by the automated imaging system arereviewed and verified manually by trained personnel before test resultsare communicated electronically via methods known in the art to aphysician. Specific cell threshold numbers can vary by specimen type andcollection method. In addition, the software can be adapted to reflectbiological, (cell shape, cell size, DNA content of the nucleus,proximity of cells to each other, cell type, etc.) or disease relateddifferences (number of loci with abnormal number, the number ofabnormalities at a locus within a single cell, relationship of anabnormality to survival or treatment response). The present embodimentscan be used on a representative sampling of area covered by cells on theslide instead of the entire area, typically this is performed by imagingmultiple fields of view or a path based on cellular density until theminimum imaged cell threshold is met. Only a subset of the rank-orderedabnormal cells can be reviewed relative to the positive/negative testthreshold as long as the clinical and disease significance is known forthe subset. Typically the subset is the most abnormal 25 or 30 cellswithin the specimens, but other subsets can be identified and utilizeddepending on the specimen source, collection method, and disease.

Yet another embodiment can be used in conjunction with tissue-basedspecimens such as those from a biopsy or surgical procedure. Inaddition, this system and method can use a companion slide that isstained with hematoxylin and eosin stain (H&E) that comes from the sametissue-based specimen. This automated method screens the entire areacovered by tissue-based specimen on the FISH prepared slide and utilizesthe DAPI-stain to identify cellular nuclei. The system then enumerateseach probe signal within the DAPI-stained region and records the copynumber of each probe identified. The software system continues itsautomated scoring of cells and chromosomal copy number within each celluntil the entire tissue-based specimen has been reviewed. The softwarethen evaluates a sub-section of the slide that contains at least 25nuclei as identified by the DAPI stain. The selection on the sub-sectionlocation is guided based upon disease indicators on the companion H&Eslide. Typically, at least two sub-sections are selected for eachspecimen. The software then categorizes each cell imaged and countedinto a category based upon the copy number of each chromosomeidentified. For instance, a normal cell with two copies of each probe3q26, 5p15, and CEN7 would be placed into a 2,2,2 category. Abnormalcells would be identified by their probe signal patterns. For instance,a cell with two copies of the CEN7 probe, 5 copies of the 3q26 probe and3 copies of the 5p15 probe would be placed in the 2,5,3 category. Onceall of the imaged cells are categorized, the number of probe signalsidentified is compared to the total number of nuclei counted by thesystem to generate a ratio of chromosomal copy number per locus versusthe number of cell counted. Once the ratio for each chromosomal loci isdetermined, the specimen can be evaluated relative to thepositive/negative disease threshold. Typically, all sub-sections of thespecimens identified with an abnormal ratio by the automated imagingsystem are reviewed and verified manually by trained personnel beforetest results are communicated to a physician. Specific sub-section sizeand number of nuclei counted can vary by specimen type and collectionmethod. In addition, the software can be adapted to reflect biological(cell shape, cell size, DNA content of the nucleus, proximity of cellsto each other, cell type, location, within the tissue, amount ofcytoplasm, etc.) or disease related differences (number of loci withabnormal number, the number of abnormalities at a locus within a singlecell, relationship of an abnormality to survival or treatment response,location of the abnormalities within the tissue-based specimen, etc.).

In yet another embodiment, the system and method can be used inconjunction with tissue-based specimens such as those from a biopsy orsurgical procedure. This automated method screens the entire areacovered by tissue-based specimen on the FISH prepared slide and utilizesthe DAPI-stain to identify cellular nuclei. The system then enumerateseach probe signal within the DAPI-stained region and records the copynumber of each probe identified. The software system continues itsautomated scoring of cells and chromosomal copy number within each celluntil the entire tissue-based specimen has been reviewed. The softwarethen evaluates a sub-section of the slides that contains at least 25nuclei as identified by the DAPI stain. Typically, at least twosub-sections are selected for each specimen. The software thencategorizes each cell imaged and counted into a category based upon thecopy number of each chromosome identified. For instance, a normal cellwith two copies of each probe 3q26, 5p15, and CEN7 would be placed intoa 2,2,2 category. Abnormal cells would be identified by their probesignal patterns. For instance, a cell with two copies of the CEN7 probe,5 copies of the 3q26 probe and 3 copies of the 5p15 probe would beplaced in the 2,5,3 category. Once all of the imaged cells arecategorized, the number of probe signals identified is compared to thetotal number of nuclei counted by the system to generate a ratio ofchromosomal copy number per locus versus the number of cell counted.Once the ratio for each chromosomal loci is determined, the specimen canbe evaluated relative to the positive/negative disease threshold.Typically, all sub-sections of the specimens identified with an abnormalratio by the automated imaging system are reviewed and verified manuallyby trained personnel before test results are communicated to aphysician. Specific sub-section size and number of nuclei counted canvary by specimen type and collection method. In addition, the softwarecan be adapted to reflect biological (cell shape, cell size, DNA contentof the nucleus, proximity of cells to each other, cell type, locationwithin the tissue, amount of cytoplasm, etc.) or disease relateddifferences (number of loci with abnormal number, the number ofabnormalities at a locus within a single cell, relationship of anabnormality to survival or treatment response, location of theabnormalities within the tissue-based specimen, etc.).

The scoring data can be analyzed by calculating the number of any one ofthe signals (e.g. 3q, 5p, or CEN7) and dividing by the total number ofnuclei scored; recording that number in the chart at the top of theScore Sheet. A result greater than 2 recorded and reported as amplifiedfar any given probe.

The scoring data is analyzed by adding the number of any one of thesignals (3q, 5p, or CEN7) and dividing by the total number of nucleiscored. A result greater than 2 can be reported as amplified for thegiven probe. Images are named by the specimen number and slide numberand saved.

In other embodiments, the present invention provides for kits for thedetection of chromosomal abnormalities at the regions disclosed. In apreferred embodiment, the kits include one or more probes to the regionsdescribed herein and any combination of the disclosed probes. The kitscan additionally include instruction materials describing how to use thekit contents in detecting the genetic alterations. The kits may alsoinclude one ore more of the following: various labels or labeling agentsto facilitate the detection of the probes, reagents for thehybridization including buffers, an interphase spread, bovine serumalbumin and other blocking agents including blocking probes, samplingdevices including fine needles, swabs, aspirators and the like, positiveand negative hybridization controls and other controls as are known inthe art.

The following illustrative explanations of the figures and relatedexamples are provided to facilitate understanding of certain terms usedfrequently herein, particularly in the examples. The explanations areprovided as a convenience and are not limitative of the invention.

EXAMPLES Example 1

FISH was performed on previously prepared thin layer, liquid-basedcytology samples (THINPREP®, Cytyc, Marlborough, Mass.). Slides weremade from THINPREP® vials and then subject to a pretreatment protocolthat includes protease digestion, formaldehyde fixation, washing, anddehydration. Hybridization was performed using a two-color multi-targetinterphase FISH probe kit (Abbot Molecular). The kit included directlylabeled probes to CEN7-aqua and to the locus of 3q26 (3q-orange) and tothe locus of 5p15 (5p-green). The cells were analyzed using fluorescencemicroscopy.

Samples had a minimum of 800 cells for analysis. Positive tests showedaneuploidy and (1) gains of either 3q copy number or 5p copy number in1.0% or more of the analyzed cells; (2) gains of only 3q copy number of0.9% or more of the analyzed cells; or (3) gains of only 5p copy numberin 0.7% or more of the analyzed cells. Negative tests showed normalploidy and (1) less than 1.0% of analyzed cells with an increase in both3q copy number and 5p copy number; (2) gains of only 3q copy number inless than 0.9% of the analyzed cells; or (3) gains of only 5p copynumber in less than 0.7% of the analyzed cells. Samples with ploidyabnormalities and/or increased 3q copy number were determined to have apoor prognosis and risk to develop more advanced cervical disease.

Results can be a diagnostic and prognostic marker for cervicaldysplasia. Samples with ploidy abnormalities and/or increased 3q copynumber and 5p copy number were determined to have a poor prognosis andrisk to develop more advanced cervical disease.

Example 2

Evaluation of FIG. 6 this specimen has revealed an abnormal copy numberof the TERC gene on 3q26 and the Cri du Chat locus 5p15. Of 1000 cellsanalyzed, 986 cells were normal while 8 were found abnormal for extracopies of TERC (3q), 2 were found abnormal for extra copies of Cri duChat (5p), and 4 were found abnormal for extra copies of TERC and Cri duChat (3q and 5p) for an abnormal cell percentage of 1.40%.

Example 3

Evaluation of FIG. 7 this specimen has revealed a normal copy number ofthe TERC gene on 3q26 and the Cri du Chat locus on 5p15. Of 1000 cellsanalyzed, 998 cells were normal while 2 were found abnormal for extracopies of TERC (3q), 0 were found abnormal for extra copies of Cri duChat (5p), and 0 were found abnormal for extra copies of TERC and Cri duChat (3q and 5p) for an abnormal cell percentage of 0.20%.

Example 4

Analysis for the Human Telomerase gene (TERC) 3q26, was performed usingFluorescent In-Situ Hybridization (FISH) with a probe specific for theTERC gene on chromosome region 3q26. In addition, a probe specific forCEN7 was applied to assess DNA ploidy. Cervical cells were fixed to aslide and hybridized with these fluorescent probes. Analysis of thehybridization was performed to assess the presence of normal andabnormal cells.

Example 5

Evaluation of FIG. 5 this specimen has revealed an abnormal copy numberof the TERC gene. Along with a representative image of cells with anabnormal copy number of TERC. Of 1030 cells analyzed, 1012 cells werenormal while 18 were found abnormal for an abnormal cell percentage of1.85%.

Example 6

Evaluation of FIG. 4 revealed a normal copy number of the TERC gene. Noamplification of the gene at 3q26 was detected and evaluation of thechromosome 7 centromere indicates a normal diploid cell. This does notrule out other abnormalities occurring at sites other than those listedabove. Along with a representative image of cells with a normal copy ofTERC. Of 800 cells analyzed, 799 cells were normal while 1 was foundabnormal for an abnormal cell percentage of 0.1%.

Example 7 Tissue FISH

FISH was performed on 4-micron thick tissue sections cut from formalinfixed paraffin-embedded (FFPE) tissue specimens. (FIGS. 8 and 9) Slideswere subject to a pre-treatment protocol that includes proteasedigestion, washing, and dehydration. Hybridization was performed using atwo-color FISH probe set containing directly labeled probes to CEN7-aquaand to the locos of 3q26 (3q-orange) (Probes obtained from AbbottMolecular). The sections were counterstained with DAPI and the cellswere analyzed using fluorescence microscopy.

A minimum of fifty cells per sample were analyzed for ploidy status.Samples were judged aneuploid if the ratio of 3q26 probe signal tonuclei within the selected cells was 2.0 or greater. Samples were judgedto have normal ploidy if the ratio of 3q26 probe signals to nucleiwithin the selected cells was less than 2. Patients with ploidyabnormalities and/or increased 3q copy number were determined at riskfor a poor prognosis and are at high risk to develop more advancedcervical disease.

Results can be a diagnostic and prognostic marker for cervicaldysplasia. Samples with ploidy abnormalities and/or increased 3q copynumber were determined at risk for poor prognosis and to developmore-advanced cervical disease.

Example 8

FISH was performed on 4-micron thick tissue sections cut from formal infixed paraffin-embedded (FFPE) tissue specimens. (FIG. 10) Slides weresubject to a pretreatment protocol that includes protease digestion,washing, and dehydration. Hybridization was performed using athree-color FISH probe set. The set included directly labeled probes toCEN7-aqua and to the locus of 3q26 (3q-orange) and to the locus of 5p15(5p-green) (probes obtained from Abbott Molecular). The sections werecounterstained with DAPI and the cells were analyzed using fluorescencemicroscopy. A minimum of fifty cells per sample were analyzed for ploidystatus.

Samples were judged to have aneuploidy if (1) the ratio of 3q26 probesignal to nuclei within the selected cells was 2.0 or greater; (2) theratio of 5p15 probe signal to nuclei within the selected cells was 2.0or greater; (3) the ratio of 3q26 and 5p15 probe signals to nucleiwithin the selected cells was 2.0 or greater. Samples were judged tohave normal ploidy if the ratio of 3q26 or 5p15 or both 3q26 & 5p15probe signals to nuclei within the selected cells was less than 2.Samples with ploidy abnormalities and/or increased 3q copy number weredetermined at risk for poor prognosis and at risk to develop moreadvanced cervical disease.

Results can be a diagnostic and prognostic marker for cervicaldysplasia. Samples with ploidy abnormalities and/or increased 3q or 5pcopy number were determined at risk for a poor prognosis and at risk todevelop more advanced cervical disease.

Example 9 Preparation of Working Reagents

To prepare 1% Formaldehyde Solution, add together: 2.7 mL 37%formaldehyde solution, 97.3 mL 1×PBS, 100 mL final volume. Mixthoroughly. Pour the solution into a Coplin jar. To prepare 2×SSC, addtogether: 100 ml 20×SSC, 900 ml dH2, 1000 ml final volume

Example 10 Ethanol Washing Solutions

Prepare dilutions of 70%, 90% using 100% ethanol and dH20. Dilutions maybe used for one week unless evaporation occurs or the solution becomesdiluted due to excessive use. Store at room temperature in cappedcontainers when not in use.

To prepare 0.4×SSC/O.3% NP-40, add together: 20 ml 2×SSC, 77.7 ml dH2O,0.3 ml NP-40, 100 ml final volume. Mix thoroughly. Discard used solutionat the end of each day. To prepare 2×SSC/0.1% NP-40, add together: 99.9ml 2×SSC, 0.1 ml NP-40. 100 ml Final Volume. Mix thoroughly. To prepare2×SSC, 900 ml dH2O, 100 ml 20×SSC, 1000 ml final volume 1×PBS 900 mldH2O, 100 ml 30×PBS, 1000 ml final volume 0.1M HCl 1 ml 1N HCl, 9 mldH2O, 100 ml final volume PEPSIN (Stock Solution) (100 MG/ML) pepsin 5g, sterile water 50 ml. Mix well. Keep on ice, make 50 μl aliquots,store at −20° C. PEPSIN (Working Solution, Prepare Fresh) 95.5 ml dH2O,0.5 ml 1M HCl. Prewarm at 37 C (approx. 15 min.). Add 20 microliter ofstock pepsin solution in clean coplan jar and add prewarmed 0.1M HCl.

Example 11 DNA Hybridization Procedure

Day One Procedure 1.

Prepared slides on THINPREP machine (Refer to THINPREP 2000 SOP); 2.Prepared and prewarm all reagents necessary; 3. Placed slides in 2×SSCfor 5 minutes at room temperature; 4. Placed slides in Protease solutionfor 10 minutes at 37° C. (Digestion step); 5. Placed slides in PhosphateBuffered Saline (PBS) 5 minutes at RT (Wash); 6. Placed slides in 1%Formaldehyde for 5 minutes at RT (Fixative); 7. Placed slides in 1×Phosphate Buffered Saline (PBS) 5 minutes at RT (Wash); 8. Placed slidesin 70% EtOH for 1 minute at RT; 9. Placed slides in 90% EtOH for 1minute at RT; 10. Placed slides in 100% EtOH for 1 minute at RT; 11.Dried slides; 12. Vortexed and centrifuged DNA probe and aliquotappropriate volume; 13. Added 10 ul of probe to each target area; 14.Covered with 22 mm square glass coverslip and seal with rubber cement;15. Denatured at 72 C for 2 min.; 16. Placed slides in humidifiedchamber and incubate at 37° C. for 14-16 hours (overnight).

Day Two Procedure.

Prepared and prewarmed wash solutions: 1. Removed slides from humidifiedchamber; 2. Gently removed rubber cement and immersed slides in 2×SSC atRT to remove coverslip; 3. Placed slides in 0.4×SSC/0.3% NP-40 for 1minute at 65° C.; 4. Removed and placed the slides in 2×SSC/0.1% NP-40at RT for 1 minute; 5. Removed and placed slides in a dark drawervertically to dry; 6. Applied one drop of DAPI/antifade solution ontothe target area and place a coverslip (22×50 mm) over DAPI solution,avoiding air bubbles; 7. Stored slides in the dark prior to signalenumeration.

Example 12 Analysis

DNA probe set and DAPI counterstain should be viewed on anepifluorescence microscope equipped with a 100-watt mercury lampequipped with the following filters; DAPI, Spectrum Aqua (chromosome 7centromere), Spectrum Orange (locus on 3q26), Spectrum Green (locus on5p15). Use the DAPI filter and a magnification of 100× and quicklyscanned sample area of patient slide to determine cell quantity andquality. Began analysis in the upper left quadrant of the target area.Scanned fields with 63× oil objective from left to right and top tobottom, without re-scanning the same areas. Count a total of 1000 cells(with 1 or 2 readers). This step can be automated for high-throughputanalysis. Began scanning, using Spectrum Orange filter. Counted cellswith 2 normal signals for the 3q26 probe and documented. Within eachfield of view scanned for 3q26, switched to Spectrum Green filter andrescanned the field for 5p15 probe signals. When encountering cell withmore than 23q26 or 5p15 signals, flipped to aqua channel and countedsignals for CEN7. Any cell with more than 23q26 or 5p15 signals isabnormal. Filled in cell counts in appropriate column on score sheet.Cells with 43q26 or 5p15, and 4 CEN7 signals were tetraploid and weredocumented appropriately. When performing this analysis method with atwo-color probe panel, the scanning, imaging, and signal documentationis modified to the reflect the actual two-color probe panel. Evaluationof specimen in FIG. 7 revealed a normal copy number of chromosomalregions 3q26 and 5p15. In the number of cells analyzed, amplification ofloci at 3q26 and 5p15 was note detected and evaluation of the chromosome7 centromere indicates a normal diploid specimen.

Example 13 Methods and Specimen Selection for Automated Analysis andScoring

A total of twenty (20) specimens were identified within theNeoDiagnostix tested population that had negative results for cytology,HPV and two-color method testing. In addition, these 20 specimens allhad 1000 total cells counted in order to minimize variation within theanalysis. The triple negative specimens were considered disease negativeand, therefore, any abnormal cells identified by FISH would beconsidered ‘false positives.’ Once the number of ‘false positive’ cellsis determined for the 20 specimens, the described BETAINV calculationcan be used to determine the threshold between normal and abnormalspecimens.

Results:

The results of FISH testing for the 20 triple negative specimens werereviewed to identify the total number of ‘false positive’ cells perspecimen at each chromosomal loci. A specimen had 5 cells identified byFISH to be abnormal across both loci, 3q26 and 5p15, the greatest numberof observed ‘false positive’ cells per specimen. This observation wasused within the BETAINV calculation to determine the threshold betweennormal and abnormal specimens 95% confidence. The total number of cellsentered into the equation was 1000. The BETAINV calculation returned athreshold value of 0.0104 (1.0%) or 10 cells out of 1000 counted cells.The threshold was also determined with 99% confidence and was equal to0.0129 (1.3%) or 13 cells out of 1000.

In addition, the results of FISH testing for the 20 triple negativespecimens were reviewed to identify the total number of ‘false positive’cells per specimen at ONLY chromosomal locus 5p15. The specimen had 3cells identified by FISH to be abnormal 5p15, the greatest number ofobserved ‘false positive’ cells per specimen. This observation was usedwithin the BETAINV calculation to determine the threshold between normaland abnormal specimens 9.5% confidence. The total number of cellsentered into the equation was 1000 from ND10107B. The BETAINVcalculation returned a threshold value of 0.0077 (0.7%) or 7 cells outof 1000 counted cells. The threshold was also determined with 99%confidence and was equal to 0.0099 (0.9%) or 9 cells out of 1000.

Using the ACMG guidelines, the threshold between, normal and abnormalspecimens was determined for the Cervical DNA Dtex diagnostic assay.With 95% confidence, the threshold was determined to be 10 cells out of1000 cells or 1.0% abnormal cells per specimen. Therefore, any specimenwith 1.0% or greater percentage of abnormal cells by FISH is abnormaland identifies a patient with a higher risk of progression. In thesecases, a POSITIVE diagnostic test result will be issued. When a specimenis found to have less than 1.0% abnormal cells per specimen, a NEGATIVEdiagnostic test report will be issued.

Example 14 Cervical FISH on Biopsy Specimens

Reagents that can be used are Sodium Thiocyanate (Kreatech LK-064A);Xylene (Mallinckrodt 8668-04, 500 ml); Proteinase, bacterial Type XXIV(Sigma P8038, 100 ml). Preparation of Working Reagents; PretreatmentSolution (1M sodium thiocyanate): Freshly made for each use: 2 g sodiumthiocyanate; 50 ml deionized water; and warmed to 80 C. Proteinase K: 25mg/ml Stock solution is used by suspending 0.025 g Proteinase K in 1 mlwater, store at −20 C up to one week. Freshly made for each use; 500 ulProteinase K stock solution; 49.5 ml 2×SSC; warmed to 45 C; 2×SSC; 100ml 20×SSC; 900 ml dH20; 1000 ml final volume. Ethanol Washing Solutions:Dilutions of 70%, 90% using 100% ethanol and dB20. Dilutions may be usedfor one week unless evaporation occurs or the solution becomes diluteddue to excessive use and stored at room temperature in capped containerswhen not in use. Probe Mix: 1×TERC in Spectrum Gold; 1×Cri du Chat inSpectrum Green; 0.1×CEP 7 in Spectrum Aqua; 2×SSC/O.1% Tween-20(pre-warm at 65 C). To prepare, add together: 20 ml 2×SSC; 77.7 ml dH2O;0.3 ml NP-40; 100 ml final volume. Mix thoroughly.

Day One Procedure:

(1) Bake 60 minutes at 65 C (2) Deparaffinize sections 2×10 min. inxylene. (3) Dehydrate in 2×5 min. in 100% EtOH. (4) Air dry. (5)Incubate slides in pre-warmed Pretreatment Solution for 8 minutes at 80C. (6) Wash 3 minutes at room temperature in de-ionized water. (7)Dehydrate slides in 70%, 90%, 100% EtOH, 1 min. each. (8) Air dry. (9)Incubate slides in 2×SSC for 5 min. (10) Incubate slides in pre-warmedProteinase K solution for 10 minutes at 45 C. [Alternative to Steps 5 to10; Incubate slides in pre-warmed Proteinase K solution for 55 minutesat 45 C. Proceed to Step 11 and continue.] (11) Rinse slides in 2×SSCfor 5 min. (12) Dehydrate slides m 70%, 90%, 100% EtOH, 1 min. each.(13) Air dry. (14) Place probe mix (briefly spin down), coverslip andseal with rubber cement. Use as small a cover glass as practical for thetissue section with proportionally sized coverslips. Note: tissuesections easily trap air bubbles; this can be minimized by pipetting theprobe mixture directly onto the section on the slide. (15) Co-denatureat 720 for 5 min. (16) Incubate at 37 C in a humidified chamberovernight.

Day Two Procedure:

(1) Pre-warm wash 2×SSC/O.1% Tween-20 at 65 C. (2) Soak slides in 2×SSCto float off cover-slip, avoid pulling off the cover-slip to minimizeshearing. (3) Wash the slides two times in 2×SSC/0.1% Tween-20 at 65 Cfor 5 minutes each. (4) Rinse slides for 1 minute at room temperature in2×SSC/0.1% NP-40. (5) Remove and place slides in a dark drawervertically to dry. (6) Coverslip with anti-fade w/DAPI (24×50 mm), storeslides at 4 C.

The foregoing description of some specific embodiments providessufficient information that others can, by applying current knowledge,readily modify or adapt for various applications such specificembodiments without departing front the generic concept, and, therefore,such adaptations and modifications should and are intended to becomprehended within the meaning and range of equivalents of thedisclosed embodiments. It is to be understood that the phraseology orterminology employed herein is for the purpose of description and not oflimitation. In the drawings and the description, there have beendisclosed exemplary embodiments and, although specific terms may havebeen employed, they are unless otherwise Stated used in a generic anddescriptive sense only and not for purposes of limitation, the scope ofthe claims therefore not being so limited. Moreover, one skilled in theart will appreciate that certain steps of the methods discussed hereinmay be sequenced in alternative order or steps may be combined.Therefore, it is intended that the appended claims not be limited to theparticular embodiment disclosed herein.

Each of the applications and patents cited in this text, as well as eachdocument or reference cited in each of the applications and patents(including during the prosecution of each issued patent; “applicationcited documents”), and each of the PCT and foreign applications orpatents corresponding to and/or claiming priority from any of theseapplications and patents, and each of the documents cited or referencedin each of the application cited documents, are hereby expresslyincorporated herein by reference. More generally, documents orreferences are cited in this text, either in a Reference List before theclaims; or in the text itself; and, each of these documents orreferences (“herein-cited references”), as well as each document orreference cited in each of the herein-cited references (including anymanufacturer's specifications, instructions, etc.), is hereby expresslyincorporated herein by reference.

REFERENCES

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What is claimed is:
 1. A method for detecting chromosomal abnormalitiesin a plurality of cells in a cervical sample, said method comprising: a)hybridizing a first nucleic acid sequence to a target nucleic acidsequence on chromosome 3q; b) hybridizing a second nucleic acid sequenceto a target nucleic acid sequence on a control chromosome; c) detectinga hybridization signal of the first and the second nucleic acidsequence, wherein the hybridization signal of said first and the secondnucleic acid sequences is indicative of chromosomal copy number forchromosome 3q and the control chromosome; d) scoring the chromosomalcopy number for chromosome 3q; and e) reporting whether the samplecontains chromosomal abnormalities, wherein a lack of chromosomalabnormalities in the sample exists when a gain of 3q copy number is inless than 0.9% of the cells.
 2. The method of claim 1, wherein thescoring of the chromosomal copy number for chromosome 3q is performed bycounting cells having 3q chromosomal abnormalities.
 3. The method ofclaim 1, wherein the scoring of the chromosomal copy number forchromosome 3q is performed by counting 3q hybridization signals.
 4. Themethod of claim 1, wherein the target nucleic acid sequence onchromosome 3q is at locus 3q26 or 3q27.
 5. The method of claim 1,wherein the target nucleic acid sequence on chromosome 3q is at locus3q26.1, 3q26.2, 3q26.31, 3q26.32, or 3q26.33.
 6. The method of claim 1,wherein the target nucleic acid sequence on chromosome 3q is the TERCgene.
 7. The method of claim 1, wherein the target nucleic acid sequenceon the control chromosome is CEN3.
 8. The method of claim 1, wherein themethod further comprises hybridizing a third nucleic acid sequence to atarget nucleic acid sequence on chromosome 5p.
 9. The method of claim 8,wherein the target nucleic acid sequence on chromosome 5p is TERT,TRIP13, or Cri du Chat locus at 5p15.2.
 10. The method of 8, wherein thetarget nucleic acid sequence on chromosome 5p is at locus 5p15, 5p15.3or 5p15.2.
 11. The method of claim 8, wherein a lack of chromosomalabnormalities in the sample exists when either (a) a gain of 3q copynumber is in less than 0.9% of the cells, or (b) a gain of 5p copynumber is in less than 0.7% of the analyzed cells.
 12. The method ofclaim 8, wherein a lack of chromosomal abnormalities in the sampleexists when (a) a gain of 3q copy number is in less than 0.9% of thecells, (b) a gain of 5p copy number is in less than 0.7% of the analyzedcells, or (c) a gain in both 3q and 5p copy number is in less than 1.0%of the analyzed cells.
 13. The method according to claim 1, wherein themethod further comprises hybridizing at least one additional nucleicacid sequence to a target nucleic acid sequence on chromosome 1q, 20q,12q, 19q, 11q, 6q, 17p, 7, 8q, 9q, 16q, 2q, 9p, 10q, or 18p.
 14. Themethod according to claim 1, wherein the method further compriseshybridizing at least one additional nucleic acid sequence to a targetnucleic acid sequence on chromosome 1q21-31, 20q12, 12q13-24, 19q13,11q21, 7q1-22, 8q24, 9q33-34, 16q23, 2q32, 9p22, 10q21-24, or 18p11. 15.The method of claim 1, wherein the hybridization signal is detected byFISH, CISH, PCR, ELISA, CGH, Array CGH or flow cytometry.
 16. The methodof claim 1, wherein the cervical sample is derived from a cervicalbiopsy, a punch biopsy, a pap smear, a thin layer cytological specimen,a thin layer suspension, fine needle aspiration, loop electrosurgicalexcision procedure (LEEP), hysterectomy, CONE biopsy, or endocervicalcurettage (ECC).
 17. The method of claim 1, wherein at least 800 cellsare examined.
 18. The method of claim 17, wherein at least 1000 cellsare examined.
 19. The method of claim 18, wherein 1000 cells areexamined.
 20. The method of claim 1, wherein the target nucleic acidsequence on a control chromosome is CEN7.
 21. The method of claim 20,wherein CEN7 is amplified.
 22. The method of claim 12, wherein thetarget nucleic acid sequence on chromosome 3q is TERC and the targetnucleic acid sequence on chromosome 5p is at locus 5p15.
 23. The methodaccording to claim 22, wherein the method further comprises hybridizinga fourth nucleic acid sequence to a target nucleic acid sequence onchromosome 20q.
 24. The method according to claim 23, wherein the targetnucleic acid sequence on the control chromosome is CEN7.
 25. The methodof claim 24, wherein CEN7 is amplified.
 26. The method of claim 22,wherein the target nucleic acid sequence on chromosome 5p is SEMA5A. 27.The method according to claim 26, wherein the method further compriseshybridizing a fourth nucleic acid sequence to a target nucleic acidsequence on chromosome 20q.
 28. The method according to claim 27,wherein the target nucleic acid sequence on the control chromosome isCEN7.
 29. The method of claim 28, wherein CEN7 is amplified.
 30. Themethod of claim 28, wherein the hybridization signals are detected usingdifferently colored fluorescently labeled probes.